Single-cell metabolite spectrum quantitative analysis platform and method based on droplet microfluidics

Abstract

The invention discloses a single-cell metabolite spectrum quantitative analysis platform and method based on droplet microfluidics, and relates to the technical field of mass spectrometry. The platform comprises a fluid pumping module, a cell oscillation module, a microfluidic mixed liquid drop generation module, a microfluidic mixed liquid drop reinjection and separation module and a mass spectrometry module. The fluid pumping module comprises a high-pressure nitrogen cylinder and a gas pressure driver; the cell oscillation module comprises a cell oscillator; the micro-fluidic mixed droplet generation module comprises a micro-fluidic droplet generation chip, a cell lysis solution, an internal standard, an oil phase, a cell suspension and a droplet collection and incubation centrifugal tube; the micro-fluidic mixed liquid drop reinjection and separation module comprises a micro-fluidic liquid drop reinjection and separation chip; and the mass spectrometry module comprises an electrospray needle and a mass spectrometer. According to the method, the single cell is captured and the internal standard substance is introduced at the same time by using droplet microfluidics, and mass spectrometry is carried out, so that high-flux single cell encapsulation and accurate quantification of metabolites are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of mass spectrometry analysis, and in particular to a single-cell metabolite mass spectrometry quantitative analysis platform and method based on droplet microfluidics. Background Art

[0002] Cells are the basic units of structure and function of organisms, and they contain a wealth of information about individual life activities. Analysis at the single-cell level can reflect the true situation of individual cells and avoid the deviation of research results caused by heterogeneity between cells. However, due to the extremely small size of cells and the dynamic changes of the types and contents of intracellular metabolites with the metabolic process in cells and the changes in the surrounding environment, rapid, in-situ, and real-time analysis of various components in cells has become a very challenging task.

[0003] Mass spectrometry is becoming increasingly popular in metabolomics research due to its high sensitivity, wide detection range, and molecular identification capabilities. Using this powerful technology to explore cellular metabolism at the single-cell level can not only understand cellular heterogeneity, but also obtain biological advantages corresponding to single cells or small cell subpopulations. The development of technologies such as high-coverage detection of intracellular metabolite molecules based on mass spectrometry and high-throughput metabolite detection of a large number of single cells have contributed to the development of single-cell metabolomics.

[0004] The current development direction of analytical chemistry is to make the analytical system simpler and more seamlessly integrated. Microfluidic chips have the advantages of integrated miniaturization, automation, high throughput and low sample requirements. Combined with mass spectrometry detection, high-throughput analysis of samples can be performed. Since the first method for manipulating single cells on microfluidic chips was published in 1997, microfluidic chips have been widely used for single-cell analysis. The quantification of metabolites is crucial to single-cell metabolomics because the concentration of metabolites directly reflects the metabolic state and regulates complex metabolic networks. At present, the quantification of single-cell metabolites by mass spectrometry is mainly achieved through external or internal standard methods, and it is not possible to perform high-throughput and absolute quantification of single-cell metabolites.

[0005] Therefore, it is an urgent problem for those skilled in the art to propose a single-cell metabolite mass spectrometry quantitative analysis platform and method based on droplet microfluidics to solve the difficulties existing in the prior art. Summary of the invention

[0006] In view of this, the present invention provides a single-cell metabolite mass spectrometry quantitative analysis platform and method based on droplet microfluidics, which achieves high-throughput single-cell encapsulation and accurate quantification of metabolites by simultaneously capturing single cells and introducing internal standards through droplet microfluidics, and performing mass spectrometry analysis.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A single-cell metabolite mass spectrometry quantitative analysis platform based on droplet microfluidics, comprising: a fluid pumping module, a cell oscillation module, a microfluidic mixed droplet generation module, a microfluidic mixed droplet re-injection and separation module and a mass spectrometry analysis module;

[0009] The fluid pumping module includes: a high-pressure nitrogen bottle and a gas pressure drive;

[0010] The cell oscillation module includes: a cell oscillator;

[0011] The microfluidic mixed droplet generation module includes: a microfluidic droplet generation chip, a cell lysate and an internal standard, an oil phase, a cell suspension, and a droplet collection and incubation centrifuge tube;

[0012] The microfluidic mixed droplet reinjection and separation module comprises: a microfluidic droplet reinjection and separation chip;

[0013] The mass spectrometry analysis module includes: an electrospray needle and a mass spectrometer;

[0014] The output end of the high-pressure nitrogen bottle is connected to the input end of the gas pressure drive;

[0015] The gas pressure driven output end is respectively connected to the cell lysate and internal standard input end, the oil phase input end and the cell suspension input end;

[0016] The cell lysate and internal standard output ends are connected to the first input end of the microfluidic droplet generation chip, the oil phase output end is connected to the second input end of the microfluidic droplet generation chip, and the cell suspension output end is connected to the third input end of the microfluidic droplet generation chip; the output end of the microfluidic droplet generation chip is connected to the input end of the droplet collection and incubation centrifuge tube;

[0017] The cell shaker is set on the cell suspension;

[0018] The output end of the droplet collection and incubation centrifuge tube is connected to the input end of the microfluidic droplet re-injection and separation chip; the output end of the microfluidic droplet re-injection and separation chip is connected to the input end of the electrospray needle;

[0019] The output of the electrospray needle was coupled to a mass spectrometer.

[0020] The above platform, optionally, the microfluidic droplet generation chip is provided with a microchannel, having a cell lysate and internal standard inlet, an oil phase inlet, a cell suspension inlet and a droplet collection hose connection port.

[0021] In the above platform, optionally, in the cell lysis solution and the internal standard, the cell lysis solution is methanol or acetonitrile; and the internal standard is 4-chlorophenylalanine.

[0022] The above platform, optionally, comprises a cell shaker that shakes the cell suspension via a shaking device to prevent cell sedimentation.

[0023] In the above platform, the electrospray needle is optionally a capillary needle with a tip, the tip is prepared by drawing or etching, and the tip is plated with a conductive metal layer, and the length of the electrospray needle is 1 to 50 cm.

[0024] A single-cell metabolite mass spectrometry quantitative analysis method based on droplet microfluidics, a single-cell metabolite mass spectrometry quantitative analysis platform based on droplet microfluidics applied to any of the above items, comprising:

[0025] S1. Use a high-pressure nitrogen bottle and gas pressure to drive the cell lysate, internal standard, oil phase and cell suspension to enter the microfluidic droplet generation chip through a plastic hose, and control the flow rates of the cell lysate, internal standard, oil phase and cell suspension respectively;

[0026] S2, the cell suspension is passed through a cell shaker to prevent cell sedimentation;

[0027] S3, in the microfluidic droplet generation chip, the droplet encapsulated single cell is a single cell in a cell suspension mixed with a cell lysate and an internal standard and then dispersed into microdroplets by an oil phase;

[0028] S4, collecting the micro-droplets into the droplet collection and incubation centrifuge tube through the droplet collection hose, and the single cells are incubated and fully lysed in the micro-droplets;

[0029] S5, the incubated and fully lysed droplets are input into the microfluidic droplet through the plastic hose and then injected into the separation chip, and the droplets are output slowly through the electrospray needle to adapt to the detection frequency of the mass spectrometer for mass spectrometry analysis;

[0030] S6. Obtain the relative concentration of single-cell metabolites through mass spectrometry analysis, thereby performing quantitative analysis of single-cell metabolites.

[0031] It can be seen from the above technical solutions that, compared with the prior art, the present invention provides a single-cell metabolite mass spectrometry quantitative analysis platform and method based on droplet microfluidics, which has the following beneficial effects: 1) The microfluidic droplet generation chip has a three-phase fluid, and uses the oil phase to disperse single cells, cell lysate and internal standards into countless mixed droplets. The outlet is collected by a hose, and the single cells are incubated and lysed, which can perform high-throughput packaging of single cells; 2) The quantitative analysis of single-cell metabolites is realized, and the microfluidic droplet reinjection and separation chip reinjects the cell lysate droplets and separates them through the oil phase to adapt to the mass spectrometry detection frequency, and the droplets are transmitted to the mass spectrometer through the spray needle for quantitative analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0033] Figure 1 An overall schematic diagram of a single-cell metabolite mass spectrometry quantitative analysis platform based on droplet microfluidics provided by the present invention;

[0034] Figure 2 A schematic diagram of the structure of a single-cell metabolite mass spectrometry quantitative analysis platform based on droplet microfluidics provided by the present invention;

[0035] Figure 3 It is a droplet cell encapsulation path diagram in the embodiment provided by the present invention;

[0036] Figure 4 It is a diagram of orderly arrangement of droplets in an embodiment provided by the present invention;

[0037] Figure 5 This is a diagram of droplet encapsulation after the cell suspension and 4-chlorophenylalanine are mixed in the embodiment provided by the present invention;

[0038] Figure 6 is a droplet reinjection path diagram in an embodiment provided by the present invention;

[0039] Figure 7 It is a total ion current diagram of a test mass spectrum when droplet microfluidics and mass spectrometry are integrated in an embodiment provided by the present invention;

[0040] Description of reference numerals:

[0041] 1 is a schematic diagram of droplet encapsulation of single cells; 2 is a microfluidic droplet generation chip; 3 is a cell lysate and an internal standard; 4 is an oil phase; 5 is a cell suspension; 6 is a cell shaker; 7 is a droplet collection and incubation centrifuge tube; 8 is a gas pressure drive; 9 is a high-pressure nitrogen bottle; 10 is a microfluidic droplet reinjection and separation chip; 11 is an electrospray needle; and 12 is a mass spectrometer. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] In this application, relational terms such as first and second, etc. are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. The terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of more restrictions, the elements defined by the sentence "comprise one..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0044] Reference Figure 1 and Figure 2 As shown, the present invention discloses a single-cell metabolite mass spectrometry quantitative analysis platform based on droplet microfluidics, including: a fluid pumping module, a cell oscillation module, a microfluidic mixed droplet generation module, a microfluidic mixed droplet re-injection and separation module and a mass spectrometry analysis module;

[0045] The fluid pumping module includes: a high-pressure nitrogen bottle 9 and a gas pressure drive 8;

[0046] The cell oscillation module includes: a cell oscillator 6;

[0047] The microfluidic mixed droplet generation module includes: a microfluidic droplet generation chip 2, a cell lysate and an internal standard 3, an oil phase 4, a cell suspension 5, and a droplet collection and incubation centrifuge tube 7;

[0048] The microfluidic mixed droplet reinjection and separation module comprises: a microfluidic droplet reinjection and separation chip 10;

[0049] The mass spectrometry analysis module includes: an electrospray needle 11 and a mass spectrometer 12;

[0050] The output end of the high-pressure nitrogen bottle 9 is connected to the input end of the gas pressure driver 8;

[0051] The output end of the gas pressure drive 8 is respectively connected to the input end of the cell lysate and internal standard 3, the input end of the oil phase 4 and the input end of the cell suspension 5;

[0052] The output end of the cell lysate and the internal standard 3 is connected to the first input end of the microfluidic droplet generation chip 2, the output end of the oil phase 4 is connected to the second input end of the microfluidic droplet generation chip 2, and the output end of the cell suspension 5 is connected to the third input end of the microfluidic droplet generation chip 2; the output end of the microfluidic droplet generation chip 2 is connected to the input end of the droplet collection and incubation centrifuge tube 7;

[0053] The cell oscillator 6 is arranged on the cell suspension 5;

[0054] The output end of the droplet collection and incubation centrifuge tube 7 is connected to the input end of the microfluidic droplet re-injection and separation chip 10; the output end of the microfluidic droplet re-injection and separation chip 10 is connected to the input end of the electrospray needle 11;

[0055] The output end of the electrospray needle 11 is coupled to a mass spectrometer 12 .

[0056] Furthermore, in the fluid pumping module, the sample injection device may be driven by gas pressure, or may be driven by a syringe pump or other liquid pumping forms.

[0057] Furthermore, the microfluidic droplet generation chip 2 is provided with a microchannel, having a cell lysate and internal standard 3 inlet, an oil phase 4 inlet, a cell suspension 5 inlet and a droplet collection hose connection port;

[0058] The inlet of the microfluidic droplet generation chip 2 is punched, and the cell lysate, internal standard 3, oil phase 4 and cell suspension 5 are input through a plastic hose embedded in the chip, and the flow rates of the three solutions are controlled separately; the droplet outlet is still transmitted through the plastic hose and collected in the droplet collection and incubation centrifuge tube 7 for incubation and lysis.

[0059] Furthermore, in the cell lysis solution and internal standard 3, the cell lysis solution is methanol, acetonitrile or other types of solutions for lysing cells; the internal standard includes 4-chlorophenylalanine or other stable compounds that do not exist in a single cell.

[0060] Furthermore, the cell shaker 6 shakes the cell suspension 5 through a shaking device to prevent cell sedimentation.

[0061] Furthermore, the electrospray needle 11 is a capillary needle with a tip, the tip is prepared by drawing or etching, and the tip is plated with a conductive metal layer, and the length of the electrospray needle 11 is 1 to 50 cm.

[0062] A single-cell metabolite mass spectrometry quantitative analysis method based on droplet microfluidics, a single-cell metabolite mass spectrometry quantitative analysis platform based on droplet microfluidics applied to any of the above items, comprising:

[0063] S1, using a high-pressure nitrogen bottle 9 and a gas pressure driver 8, driving the cell lysate, the internal standard 3, the oil phase 4 and the cell suspension 5 to be input into the microfluidic droplet generation chip 2 through a plastic hose, and controlling the flow rates of the cell lysate, the internal standard 3, the oil phase 4 and the cell suspension 5 respectively;

[0064] S2, the cell suspension 5 passes through a cell shaker 6 to prevent cell sedimentation;

[0065] S3, in the microfluidic droplet generation chip 2, the droplet-encapsulated single cell 1 is a single cell in a cell suspension 5 mixed with a cell lysate and an internal standard 3 and then dispersed into microdroplets by an oil phase 4;

[0066] S4, collecting the micro-droplets into the droplet collection and incubation centrifuge tube 7 through the droplet collection hose, and the single cells are incubated and fully lysed in the micro-droplets;

[0067] S5, the incubated and fully lysed droplets are input into the microfluidic droplet through the plastic hose and then injected into the separation chip 10, and the droplets are output slowly through the electrospray needle 11 to adapt to the detection frequency of the mass spectrometer 12 for mass spectrometry analysis;

[0068] S6. Obtain the relative concentration of single-cell metabolites through mass spectrometry analysis, thereby performing quantitative analysis of single-cell metabolites.

[0069] In a specific embodiment, referring to Figure 3 , Figure 4 and Figure 5 , 4-chlorophenylalanine standard solution and cell suspension were introduced into the two inlets of the Y-shaped channel as two dispersed phases, and the two aqueous phases flowed out simultaneously through the gas-driven pump. It can be seen that the two phases were separated and moved to the right together. Figure 3 The complete cell swimming path can be observed in the middle af; then the oil phase at the intersection simultaneously shears the two dispersed phase mixture up and down to obtain mixed droplets encapsulating single cells. The droplets can be seen arranged neatly in the observation cabin after the droplets are generated.

[0070] Reference Figure 6 The collected droplets are passed into the microfluidic droplet and then injected into the water phase inlet of the separation chip. Similarly, the gas-driven pump is used to separate the continuous droplets in the dispersed phase, so that the droplet frequency adapts to the mass spectrometry frequency, and flows through the electrospray needle, enters the electrospray mass spectrometer and is analyzed.

[0071] Reference Figure 7 As shown, the measured peak intensity of single-cell metabolites is compared with the peak intensity of a certain concentration of internal standard presented in the mass spectrum to obtain the relative concentration of single-cell metabolites, thereby performing quantitative analysis of single-cell metabolites.

[0072] Each embodiment in this specification is described in a progressive manner, and the same or similar parts between the embodiments can refer to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the system or system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can refer to the partial description of the method embodiment. The system and system embodiments described above are merely schematic, wherein the units described as separate components may or may not be physically separated, and the components displayed as units may or may not be physical units, that is, they may be located in one place, or they may be distributed on multiple network units. Some or all of the modules may be selected according to actual needs to achieve the purpose of the scheme of this embodiment. Ordinary technicians in this field can understand and implement it without creative work.

[0073] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A single-cell metabolite mass spectrometry quantitative analysis platform based on droplet microfluidics, characterized in that: include: Fluid pumping module, cell oscillation module, microfluidic mixed droplet generation module, microfluidic mixed droplet re-injection and separation module and mass spectrometry analysis module; The fluid pumping module comprises: a high-pressure nitrogen bottle (9) and a gas pressure drive (8); The cell oscillation module includes: a cell oscillator (6); The microfluidic mixed droplet generation module comprises: a microfluidic droplet generation chip (2), a cell lysate and an internal standard (3), an oil phase (4), a cell suspension (5), and a droplet collection and incubation centrifuge tube (7); The microfluidic mixed droplet re-injection and separation module comprises: a microfluidic droplet re-injection and separation chip (10); The mass spectrometry analysis module comprises: an electrospray needle (11) and a mass spectrometer (12); The output end of the high-pressure nitrogen bottle (9) is connected to the input end of the gas pressure driver (8); The output end of the gas pressure drive (8) is respectively connected to the input end of the cell lysate and internal standard (3), the input end of the oil phase (4) and the input end of the cell suspension (5); The output end of the cell lysate and internal standard (3) is connected to the first input end of the microfluidic droplet generation chip (2), the output end of the oil phase (4) is connected to the second input end of the microfluidic droplet generation chip (2), and the output end of the cell suspension (5) is connected to the third input end of the microfluidic droplet generation chip (2); the output end of the microfluidic droplet generation chip (2) is connected to the input end of the droplet collection and incubation centrifuge tube (7); A cell oscillator (6) is disposed on the cell suspension (5); The output end of the droplet collection and incubation centrifuge tube (7) is connected to the input end of the microfluidic droplet re-injection and separation chip (10); the output end of the microfluidic droplet re-injection and separation chip (10) is connected to the input end of the electrospray needle (11); The output end of the electrospray needle (11) is coupled to a mass spectrometer (12).

2. The single-cell metabolite mass spectrometry quantitative analysis platform based on droplet microfluidics according to claim 1, characterized in that: The microfluidic droplet generation chip (2) is provided with a microchannel, having a cell lysate and internal standard (3) inlet, an oil phase (4) inlet, a cell suspension (5) inlet and a droplet collection hose connection port.

3. The single-cell metabolite mass spectrometry quantitative analysis platform based on droplet microfluidics according to claim 1, characterized in that: In the cell lysis buffer and internal standard (3), the cell lysis buffer is methanol or acetonitrile; the internal standard is 4-chlorophenylalanine.

4. The single-cell metabolite mass spectrometry quantitative analysis platform based on droplet microfluidics according to claim 1, characterized in that: The cell shaker (6) shakes the cell suspension (5) through a shaking device to prevent cell sedimentation.

5. The single-cell metabolite mass spectrometry quantitative analysis platform based on droplet microfluidics according to claim 1, characterized in that: The electrospray needle (11) is a capillary needle with a tip, the tip is prepared by drawing or etching, and the tip is plated with a conductive metal layer. The length of the electrospray needle (11) is 1 to 50 cm.

6. A method for quantitative analysis of single-cell metabolites by mass spectrometry based on droplet microfluidics, applied to a single-cell metabolites by mass spectrometry quantitative analysis platform based on droplet microfluidics according to any one of claims 1 to 5, comprising: S1, using a high-pressure nitrogen bottle (9) and a gas pressure driver (8), driving the cell lysate and the internal standard (3), the oil phase (4) and the cell suspension (5) to be input into the microfluidic droplet generation chip (2) through a plastic hose, and controlling the flow rates of the cell lysate and the internal standard (3), the oil phase (4) and the cell suspension (5) respectively; S2, the cell suspension (5) passes through a cell shaker (6) to prevent cell sedimentation; S3, in the microfluidic droplet generation chip (2), the droplet-encapsulated single cell (1) is a single cell in a cell suspension (5) that is mixed with a cell lysate and an internal standard (3) and then dispersed into microdroplets by an oil phase (4); S4, collecting the micro-droplets into the droplet collection and incubation centrifuge tube (7) through the droplet collection hose, and the single cells are incubated and fully lysed in the micro-droplets; S5, the incubated and fully lysed droplets are input into the microfluidic droplet control chip (10) through a plastic hose, and the droplets are slowly output through an electrospray needle (11) to adapt to the detection frequency of a mass spectrometer (12) for mass spectrometry analysis; S6. Obtain the relative concentration of single-cell metabolites through mass spectrometry analysis, thereby performing quantitative analysis of single-cell metabolites.

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